pET22b_PstS_3
(Plasmid #78200)

• Purpose: Expression of double-cysteine variant of mature phosphate binding protein, PstS, which also has a mutation to weaken phosphate binding, as scaffold for phosphate biosensor
• Depositing Lab: Martin Webb
• Publication: Solscheid et al Biochemistry. 2015 Aug 18;54(32):5054-62. doi: 10.1021/acs.biochem.5b00449. Epub 2015 Aug 4.

Backbone

• Vector backbone: pET22b
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: use BL21 (DE3) for protein expression
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: pstS (A17C,I76G,A197C)
• Species: E. coli
• Mutation: A17C,I76G,A197C (please see depositor comment below)
• Promoter: T7

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: NdeI (unknown if destroyed)
• 3′ cloning site: XhoI (unknown if destroyed)
• 5′ sequencing primer: T7
• 3′ sequencing primer: T7 terminal

Resource Information

• Supplemental Documents:
  • pET22b_PstS_3 protocol
  • pET22b_PstS_3 information

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

Please note- The numbering system used was for the mature natural wild-type PstS protein. The plasmid produces an extra N-terminal methionine so all mutations will be off by 1nt (e.g., A197C =A198C)

PstS ORF encoding the mature E. coli (A17C, I76G, A197C)PstS protein (PBPw) containing a stop codon at the end of the gene in pET22b (inserted between Nde1 and Xho1 sites in the MCS).

Expression produces MEA at the N-terminus of the mature PstS: So there is an extra M at the N-terminus over the natural, mature PstS. A stop codon was also added at the end of the sequence BEFORE the plasmid-derived His tag. This gives PLY at the C-terminus at the C-terminus of the expressed protein, as in the natural PstS. The amino-acid numbering has been left as based on positions in the natural, mature protein.

Usage
The expressed protein is a weak-binding variant of E. coli phosphate binding protein, PstS. It contains two cysteines (A17C, A197C) and a mutation to weaken binding (I76G). The gene encodes the mature protein without the N-terminal signal peptide, but with an N-terminal methionine. Subsequently the cysteines are labeled with 2 6-iodoactemidotetramethylrhodamines per molecule. This adduct is then used as a phosphate biosensor, having up to 9-fold fluorescence intensity increase on binding inorganic phosphate, with a low dissociation constant so inorganic phosphate can be measured in the tens of micromolar range.